Spatially resolved proteomic map shows that extracellular matrix regulates epidermal growth

Human skin comprises stratified squamous epithelium and dermis with various stromal cells and the extracellular matrix (ECM). The basement membrane (BM), a thin layer at the top of the dermis, serves as a unique niche for determining the fate of epidermal stem cells (EpSCs) by transmitting physical and biochemical signals to establish epidermal cell polarity and maintain the hierarchical structure and function of skin tissue. However, how stem cell niches maintain tissue homeostasis and control wound healing by regulating the behavior of EpSCs is still not completely understood. In this study, a hierarchical skin proteome map is constructed using spatial quantitative proteomics combined with decellularization, laser capture microdissection, and mass spectrometry. The specific functions of different structures of normal native skin tissues or tissues with a dermatologic disease are analyzed in situ. Transforming growth factor-beta (TGFβ)-induced protein ig-h3 (TGFBI), an ECM glycoprotein, in the BM is identified that could enhance the growth and function of EpSCs and promote wound healing. Our results provide insights into the way in which ECM proteins facilitate the growth and function of EpSCs as part of an important niche. The results may benefit the clinical treatment of skin ulcers or diseases with refractory lesions that involve epidermal cell dysfunction and re-epithelialization block in the future.

14. Line 83-84: there are several phosphorylation sites for β-catenin; some are for degradation, e.g. S45, S33/37/T41; some are for activity and nucleus accumulation, e.g. S552. It should be defined clearly. The references should be updated to more recent articles.
15. Line 290: "…syphilis infection destroyed epidermal development and….". Epidermal development usually indicates the developmental process during embryogenesis. It would be more appropriate to use "epidermal homeostasis" or "epidermal stratification".
16. Line 343-350: No description or data interpretation on the effects of XAV939 in TGFBI-treated organoids. It remains unclear the relationship between Wnt signaling and TGFBI.
17. Line 363-364: Aren't MMP proteins involved in cell migration rather than cell proliferation? Please confirm it.
18. English writing needs to be edited carefully. For examples: -Line 313: "TGFBI expression could be induced with TGFβ1 and was secreted in the extracellular space in fibroblasts." should be "TGFBI expression could be induced by the treatment of TGFβ1 and was secreted in the extracellular space in between fibroblasts". -Line 330: According to the diagram shown in Fig.4j, "EpSC-derived hiPSCs (EpSC-hiPSCs)" is not an accurate term; it should be "EpSCs derived from hiPSCs" or "hiPSC-derived EpSCs".
Reviewer #2 (Remarks to the Author): I was asked to evaluate the proteomics portion of this work. The authors did an excellent job of designing the study, from the information I had available to me. All the standard tools were used for processing and analysis, and this work was done at a very high level. The authors are to be commended for doing replicate injections of replicate samples for each skin layer; that gives the absolute best/most reliable quantification in this type of experiment.
If the authors truly did not randomize their samples at all, though, that is somewhat problematic. Under these circumstances, it is difficult to conclude whether any significant changes are due to real, biological differences or due to instrument drift over time or other possible confounding effects. Also, I was not able to examine the raw data in the ProteomeXchange submission because I did not have the login information.
From the information available to me, this work appears to be very good. However, I would like to examine the data in ProteomeXchange and also have the authors address the issue of sample randomization.
Reviewer #3 (Remarks to the Author): This data-rich and generally well-written manuscript describes use of tissue-specific proteome and transcriptome analysis. In general, the data are convincing. However, the results are incompletely described, and in particular the figure legends contain insufficient information for the reader to be able to interpret them. Pertinent prior studies are not incorporated in the interpretation or cited. The overall meaning of the results are also not described adequately.
1. l. 179-181 and elsewhere. In many places in the manuscript, varied abundance of proteins in different tissue regions is referred to as upregulation or downregulation. This terminology is incorrect, because (as exemplified by keratins in the epidermis and collagen I in the dermis) many proteins accumulate during development. Their levels do not necessarily correlate with the relative mRNA levels or ONGOING protein production in the tissue region. For example, the stratum corneum lacks nuclei and likely has little new mRNA and protein production; most of the proteins present was produced during the cells' development in the stratum spinosum and granulosum. The proteins are also subject to differential degradation and modification. Therefore it would more appropriate to refer to differences in levels of protein abundance rather than upregulation or downregulation. 2. l. 197. Does it make sense that proteins associated with neuronal development are present in high levels in the GS layer? It would be better to examine the underlying functions of the proteins, which in this tissue would be expected to have little role in neuronal development.
l. 362 and Fig. 6. The use of the term 'epithelial tongue' would be confusing to most readers. Alternative wording should be used.
l. 486. The equipment used for laser dissection is not described.
The manuscript would benefit from a figure showing a model of how TGFBI expression is proposed to be related to epidermal development, EpSC proliferation, and expression of the proteins involved in differentiation of the layers. As it stands, the overall findings are unclear.
In general, the figure legends should be sufficiently detailed so that the reader does not have to refer to the body of the manuscript to easily understand the figure. That is not the case for any of the figures. The figures would also benefit from labeling and arrows showing key features. Specific comments are provided below. legend. The first sentence should state "in normal human skin" or something similar. The images in 1a should have labeling for the epidermis, basement membrane and dermis, and the legend should describe the coloration of each specific label and the DAPI staining. In 1c, "Spatially" should be changed to "Spatial", and the validation description should be expanded somewhat. In 1d legend, "Number of proteins detected" is suggested. In 1g, the meaning of "summed intensities of the proteins of interest by the summed intensities of all proteins" is unclear, in that the summed values for each region do not approach 100 (particularly for the basement membrane). Fig. 2. For the 2a legend, it should be indicated that each column within the skin regions corresponds to a different human skin specimen (if that is the case). Why is the number variable? Perhaps dashed lines could be drawn at the upper and lower boundaries to more clearly associate the regions of the heat map with the descriptions on the right side. In 2c, the EGFR and COL4AJ specimens appear to be IHC rather than immunofluorescence, and the color difference between the specific staining and nonspecific stain (hematoxylin?) is not sufficient to discern. These should be redone. As for 1a, 2c should be more thoroughly described in the legend; perhaps arrows could be added to indicate areas of specific staining. Fig. 3. In 3a, what is the staining shown under "Treponema pallidum"? The staining method is not described. Also, the coloration in the lower right panel of 3a does not match that of the low magnification view. Scale bars should be used consistently and labeled. In 3b legend, SSP-GS should be described as the stratum granulosum-spinosum layers rather than stratum corneum. For 3d legend, the term "gradually" does not fit here. In 3e, the NC and SSP groups should be more clearly separated in the figure. Fig. 4. The title sentence should indicate that the data in this figure is based primarily on RNA-seq analysis, as opposed to protein analysis in the preceding figures. In 4a, the coloration and the meaning of the dashed line should be described. In 4b legend, it should be stated that these are in vitro cultures of EpSCs. The treatment interval (hours) should be indicated. For 4h, the x axis should be labeled, and a more standard y axis (rather than e5) should be used. Fig. 4j is unclear and should be omitted.
l. 248 and elsewhere. expression rather than expressions.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In this manuscript, authors used laser capture microdissection and mass spectrometry to construct the spatial proteome of stratified human skin. By comparing normal skin to the skin tissues with secondary syphilis (SSP), they identified TGFBI as a key factor that enhances the growth of epidermal stem cells (EpSC) and the process of wound healing. This manuscript provides a vast amount of proteomic data which would be useful for future research; however, unclear interpretation and data presentation lead to inadequate conclusion and would cause misleading to the readers. Although their data are potentially interesting, appropriate editing on their interpretation, English writing and additional data analysis are essential before it can be recommended for publication. Several concerns are listed as follows: 1. Overall, the presentation of figures should be re-organized for a better appreciation; e.g Fig. 3, sub-figures are not arranged in order. Fig.S6k was mentioned in the main text before Fig.6i, j; thus, the sequence should be switched accordingly. Moreover, quantification data for several immunostaining analyses are missing, e.g. Fig.4g, 4k, 4o, 5a, 5c, 5d and 7c.
Answer: Many thanks for the editor's suggestion.
In the revised manuscript, we have re-organized Fig. 3 and Fig. S6 to make the sub-figures in order. Also, we have added the quantified data for immunostaining analyses in Fig 4f, 4h, 5a, 5c, 5d, 7b and 7c. 2   2. In addition, although authors tended to link TGFBI to Wnt signaling activation, the information about how TGFBI activates Wnt signaling is very limited. The effects of Wnt signaling inhibitor, XAV939, on cell proliferation and protein expression could be independent of TGFBI. Authors will need to show direct evidence to demonstrate that TGFBI-mediated regulation on EpSCs depends on Wnt signaling.
Answer: Many thanks for the reviewer's suggestion.
In the revised version, we performed experiments to provide direct evidence and the explanation for the correlation between TGFBI and Wnt signaling on EpSCs.
First, we found clues through the interaction between TGFBI and proteins identified in BL.
As shown in Fig. S4f, TGFBI interacted with several Wnt pathway related proteins, including ITGB1 and ITGA3. We also proved that the expression of ITGB1 increased after TGFBI treatment for EpSCs (Fig. 5b). Then, we found that with TGFBI treatment, the expression of transcription factor (LEF-1) of Wnt signal pathway increased (Fig. 4k). Besides, total β-catenin expression and its expression in nucleus increased (Fig.4l). It is known that a small part of β-catenin is phosphorylated (such as S33/37/T41) under natural conditions (Cell. 2002, 108(6):837-47.). Phosphorylated β-catenin and GSK3β can form complexes and be degraded by ubiquitination. Our results showed that after TGFBI treatment, the total GSK3β expression decreased, phosphorylated GSK3β increased, which may lead to a decrease of β-catenin degradation (Fig. 4k and Fig. S6i). When XAV939, which can promote β-catenin degradation, was added in the EpSCs, even with the treatment of TGFBI, the degradation of β-catenin was not improved (Fig. 4k,l and Fig. S6h). In addition, with XAV939 and TGFBI treatment, the expression of total GSK3β did not decrease, phosphorylated β-catenin (S33/37/T41) increased (Fig. 4k), and the expression of β-catenin in the nucleus decreased (Fig. 4l). These results showed the regulatory relationship between TGFBI and Wnt pathway.
On the other hand, we found that the proliferation and stemness associated proteins of EpSCs increased after TGFBI treatment. When we added siTGFBI, the proliferation and stemness ability 6 of EpSCs decreased slightly in EpSCs, while the expression level of EpSCs function related proteins (PCNA and total β-catenin) recovered after TGFBI treatment (Fig. S6j). These results indicated that TGFBI can regulate EpSCs' function. After adding XAV939 or adding TGFBI after adding XAV939, we found that while Wnt signals were not activated, the function associated markers of EpSCs could not be restored. The above results provide the evidence for TGFBI-mediated regulation on EpSCs depends on Wnt signaling.    Answer: Many thanks for the reviewer's suggestion.
According to our previous experiments, TGFBI is expressed in dermal fibroblasts after TGFβ stimulation, while TGFBI is almost not expressed in epidermal cells ( Figure 1). Therefore, there should be a large amount of TGFBI in the dermis. In this study, the TGFBI protein has been identified with a high intensity in basement membrane by LC-MS/MS method; further, through the immunochemical staining verification, TGFBI is proved located in the basement membrane (Fig. 4a and Fig. S6a). It is speculated that the TGFBI protein is secreted by fibroblasts first and then located in the basement membrane. As the structure of pathological section varies with different tissue part, the expression level of TGFBI in different sections may differ slightly. To make the results more clear, we have updated the staining pictures in Fig. 3f. As shown in Fig. 3f and Fig. 4a, there is a large amount of TGFBI in the basement membrane (dotted line) and superficial dermis of skin tissues. In addition, the dermis contains blood vessels and TGFBI also locates in basement membrane under the vascular endothelial cells. Therefore, it seems that more TGFBI is expressed on the pathological section of dermis with more blood vessels.   Answer: Many thanks for the reviewer's suggestion.
In order to demonstrate the activation of Wnt/β-catenin, we added the western blot results of total GSK3β and the levels of p-β-catenin (S33/37/T41) in Fig. 4k. The expression of total β-catenin have been provided in Fig. 4k; and the levels of cytoplasmic / nuclear fraction of β-catenin have been provided in Fig. 4l. In addition, we added the results of the increased expression of transcription factor (LEF-1) of Wnt signal pathway after TGFBI treatment (Fig.4k).
Further, we found that knockdown of TGFBI can lead to the decrease of β-catenin; while knockdown of TGFBI with TGFBI supplement, the β-catenin restore to the normal level (Fig. S6j).
These results indicated the regulated relationship between TGFBI and Wnt/β-catenin signaling.      8. Fig.5c: Some of immunofluorescence staining images are over-exposed, e.g. Fig.5c middle panel for TGFBI. Also, CLDN1 seems not located at cell junction in the cells treated with TGFBI for 7 days.
Answer: Many thanks for the reviewer's suggestion.
The images in Fig. 5 are the final result of overlaying the images from different layers of the 3D spheroids, thus, some images look a little over-exposed.
The following Figure 3 show the images of different layers of the 3D spheroids treated with TGFBI, which are corresponding to the middle panel in Fig. 5c. It can be found that CLDN1 is expressed both in cell membrane and nucleus, and it seems that the high level expressions of CLDN1 in nucleus could cover its expression on cell membrane. Answer: Many thanks for the reviewer's suggestion.
In the revised manuscript, we have provided the WB data of TGFBI knockdown in mouse skin tissues in Fig.S7a. In Fig.S7b and S7c, we have added the wound healing images of the mouse skin tissues and the quantitative analysis of wound healing area. Answer: Many thanks for the reviewer's suggestion.
In Fig.6, we want to show the proliferation and tongue sites at day 7 and day 14. In the previous manuscript, we made a mistake in the description of figure legend. Fig. 6d and 6e represent the 14th day after wound healing, not the 7th day. Therefore, the epidermis had basically completed re-epidermization on the day 14, and the epithelial tongue could not be observed.
In the revised manuscript, we have corrected this mistake in figure legend of Fig. 6, and also revised the marker label of the lower panel of Fig. 6e to "PCNA and MMP7". Did authors mean to say "EpSCs undergo epithelial-mesenchymal transition"? or did authors identify "excess production of ECM proteins by SSP-EpSCs"? Please explain it.
Answer: Many thanks for the reviewer's suggestion.
We compared the EpSCs isolated from the skin tissues of SSP patients and normal people.
By analysis of the differential expressed proteins, there were 84 ECM proteins that expressed with higher abundance in SSP patients compared with normal people, including 3 collagens, 15 glycoproteins, 1 proteoglycans, 20 ECM affiliated proteins, 34 ECM regulated proteins, and 11 secretory factors (Figure 4a). Most of these ECMs are involved in the biological processes of ECM assembly, proteolysis, epidermal cell differentiation, response to wound healing, and regulation of fibroblast migration (Figure 4b). Among them, COL18A1, LAMA5, LAMA1, and TGFβ were related to epithelial-mesenchymal transition. Therefore, from the perspective of ECM expression and cell morphology, EpSCs isolated from patient skin tissues are more difficult to maintain their stemness and lead to over differentiate.   14. Line 83-84: there are several phosphorylation sites for β-catenin; some are for degradation, e.g.
S45, S33/37/T41; some are for activity and nucleus accumulation, e.g. S552. It should be defined clearly. The references should be updated to more recent articles.
Answer: Many thanks for the reviewer's suggestion.
We have tested the phosphorylation site S33/37/T41 on β-catenin, and found it was downregualted after TGFBI treatment, and this result has been provided in Fig. 4k. Also, we have added the descriptions of phosphorylation sites for β-catenin and updated the references in introduction. Answer: Many thanks for the reviewer's suggestion.
We revised the statement of "epidermal development" to "epidermal homeostasis".
16. Line 343-350: No description or data interpretation on the effects of XAV939 in TGFBI-treated organoids. It remains unclear the relationship between Wnt signaling and TGFBI.
Answer: Many thanks for the reviewer's suggestion.
We added the description to interpret the effects of XAV939 in TGFBI-treated organoids, as follows: "Furthermore, the expression of maturation marker KRT10 was upregulated in 3D EpSC cultures after XAV939 treament, even with TGFBI added (Fig. 5a), indicating that TGFBI can promote the EpSC proliferation but does not show the ability to promote EpSC differentiation." 17. Line 363-364: Aren't MMP proteins involved in cell migration rather than cell proliferation?
Please confirm it.
Answer: Many thanks for the reviewer's suggestion.
MMP proteins can degrade ECM and expand the space required for cell migration. We revised the statement of "cell proliferation" to "cell migration".

English writing needs to be edited carefully. For examples:
-Line 313: "TGFBI expression could be induced with TGFβ1 and was secreted in the extracellular space in fibroblasts." should be "TGFBI expression could be induced by the treatment of TGFβ1 and was secreted in the extracellular space in between fibroblasts".
Answer: Many thanks for the reviewer's suggestion.
The sentence "TGFBI expression could be induced with TGFβ1 and was secreted in the extracellular space in fibroblasts." has been revised to "TGFBI expression could be induced by the treatment of TGFβ1 and was secreted in the extracellular space between fibroblasts ( Supplementary Fig. 6c-e)." The description "EpSC-derived hiPSCs (EpSC-hiPSCs)" has been revised to "hiPSC-derived EpSCs" Besides, we have checked the manuscript detailedly and corrected several language errors.
Also, the manuscript has been revised by a native English speaker.
Reviewer #2 (Remarks to the Author): I was asked to evaluate the proteomics portion of this work. The authors did an excellent job of designing the study, from the information I had available to me. All the standard tools were used for processing and analysis, and this work was done at a very high level. The authors are to be commended for doing replicate injections of replicate samples for each skin layer; that gives the absolute best/most reliable quantification in this type of experiment.
If the authors truly did not randomize their samples at all, though, that is somewhat problematic.
Under these circumstances, it is difficult to conclude whether any significant changes are due to real, biological differences or due to instrument drift over time or other possible confounding effects. Also, I was not able to examine the raw data in the ProteomeXchange submission because I did not have the login information.
From the information available to me, this work appears to be very good. However, I would like to examine the data in ProteomeXchange and also have the authors address the issue of sample randomization.
Answer: Many thanks for the reviewer's approval of our work.
This study includes two groups, the secondary syphilis patients group and control donors group. Five patients and the matched controls that conformed to the inclusion criteria were randomly sampled from the biobank of the Department of Dermatology and Venereology, Peking Union Medical College Hospital. Thus, the tissue samples used in this study were randomly selected.
In "Life sciences study design" section in the "Reporting summary" document, we have filled the following information in "Randomization" column "No randomization took place during processing or analyses of tissue and cells." This means that the skin tissues from different patients or controls were processed separately; add the skin tissues from different individual didn't mixed.
To avoid possible misunderstanding, we have revised the "Reporting summary" document to make the meaning more clear.
All proteomics raw data have been deposited to the ProteomeXchange Consortium via the iProX partner repository with the dataset identifier PXD027093. In the peer review process, it can be access via this link (https://www.iprox.cn/page/PSV023.html;?url=16254639404822ohA, Xp8E); and the dataset will be public available when this paper is published.

Reviewer #3 (Remarks to the Author):
This data-rich and generally well-written manuscript describes use of tissue-specific proteome and transcriptome analysis. In general, the data are convincing. However, the results are incompletely described, and in particular the figure legends contain insufficient information for the reader to be able to interpret them. Pertinent prior studies are not incorporated in the interpretation or cited.
The overall meaning of the results are also not described adequately. 1. l. 179-181 and elsewhere. In many places in the manuscript, varied abundance of proteins in different tissue regions is referred to as upregulation or downregulation. This terminology is incorrect, because (as exemplified by keratins in the epidermis and collagen I in the dermis) many proteins accumulate during development. Their levels do not necessarily correlate with the relative mRNA levels or ONGOING protein production in the tissue region. For example, the stratum corneum lacks nuclei and likely has little new mRNA and protein production; most of the proteins present was produced during the cells' development in the stratum spinosum and granulosum. The proteins are also subject to differential degradation and modification. Therefore it would more appropriate to refer to differences in levels of protein abundance rather than upregulation or downregulation.
Answer: Many thanks for the reviewer's suggestion.
We have taken the reviewer's suggestion and revised the description of varied abundance of proteins (lines 179-181 in the previous version), as follows: "We analyzed the proteins with different abundance expression trends in epidermal cells from the BL to the GS and SC. The proteins with increased abundance were enriched in…The proteins with decreased abundance from the BL…". The similar descriptions in other parts of this article have also been revised.
The descriptions of "upregulated/downregulated" kept in the text are specifically referred to the proteins or genes that are significantly differentially expressed in two compared group by statistical analysis.
2. l. 197. Does it make sense that proteins associated with neuronal development are present in high levels in the GS layer? It would be better to examine the underlying functions of the proteins, which in this tissue would be expected to have little role in neuronal development.
Answer: Many thanks for the reviewer's suggestion.
We have modified the sense and removed the description of proteins associated with neuronal development in the text.
l. 362 and Fig. 6. The use of the term 'epithelial tongue' would be confusing to most readers.
Alternative wording should be used.
Answer: Many thanks for the reviewer's suggestion.
In the revised manuscript, we have revised the term "epithelial tongue" as "wound edge".
l. 486. The equipment used for laser dissection is not described.
we added the equipment used for laser dissection.
Answer: Many thanks for the reviewer's suggestion.
We added the equipment used for laser dissection.
The equipment used for laser dissection has been provided at the beginning of "LCM of eight layers of skin samples" section in Methods, as follows: "LCM was performed using a laser microdissection system from Molecular Machines and Industries (MMI CellCut Laser Microdissection, Eching, Germany)".

The manuscript would benefit from a figure showing a model of how TGFBI expression is
proposed to be related to epidermal development, EpSC proliferation, and expression of the proteins involved in differentiation of the layers. As it stands, the overall findings are unclear.
Answer: Many thanks for the reviewer's suggestion.
In the revised manuscript, we have added the supplementary Fig. S8 to show the schematic diagram of TGFBI promoting skin re-epidermization during wound healing through enhancing the growth of EpSCs. Specific comments are provided below.
Answer: Many thanks for the reviewer's suggestion.
In the revised manuscript, figure legends have been extended with more details. for the epidermis, basement membrane and dermis, and the legend should describe the coloration of each specific label and the DAPI staining. In 1c, "Spatially" should be changed to "Spatial", and the validation description should be expanded somewhat. In 1d legend, "Number of proteins detected" is suggested. In 1g, the meaning of "summed intensities of the proteins of interest by the summed intensities of all proteins" is unclear, in that the summed values for each region do not approach 100 (particularly for the basement membrane).
Answer: Many thanks for the reviewer's suggestion.
We revised the legend of Fig. 1 and added more labels and explanation in figure.
The title of Fig. 1 has been revised to "Quantitative proteome profiling of spatially distinct protein signatures in normal human skin." The epidermis, basement membrane, and dermis of the images in 1a have been labeled, and the coloration of each specific label and the DAPI staining has been described in the legend.
The label "Spatially proteome" has been modified to "Spatial proteome" in Fig. 1c.
The legend of Fig. 1d has been revised to "Number of proteins identified in SC, GS, BL, BM, SD, and DD." In 1g, the percentage was calculated by dividing the summed intensities of the proteins of interest (keratins, collagens, glycoproteins, proteoglycans, ECM regulators, ECM-affiliated proteins, or secreted proteins) by the summed intensities of all proteins identified in specific skin layer. Except for the seven kinds of proteins counted in Fig. 1g, there are many other proteins identified in each skin layer. Thus, the summed percentages of these seven kinds of proteins do not approach 100.
Taking the stratum corneum (SC) as an example, a total of 4686 proteins identified in SC with the sum protein intensities of 1.65E+09. Within the 4686 proteins, 50 keratins, 28 collagens, 82 glycoproteins, 15 proteoglycans, 93 ECM regulators, 39 ECM-affiliated proteins, and 40 secreted proteins were identified. The summed intensities of the keratins were 4.51E+08, then, the percentage for keratins in SC was calculated as: 4.51E+08/1.65E+09*100%=27.31%. Perhaps dashed lines could be drawn at the upper and lower boundaries to more clearly associate the regions of the heat map with the descriptions on the right side. In 2c, the EGFR and COL4AJ specimens appear to be IHC rather than immunofluorescence, and the color difference between the specific staining and nonspecific stain (hematoxylin?) is not sufficient to discern. These should be redone. As for 1a, 2c should be more thoroughly described in the legend; perhaps arrows could be added to indicate areas of specific staining.
Answer: Many thanks for the reviewer's suggestion.
In Fig. 2a legend, we have indicated the meaning of each column as follows: "Each column within the skin regions corresponds to a different human skin specimen." The skin tissues from five healthy donors were used as control groups. We performed the laser capture microdissection (LCM) on the frozen embedded sections to get different skin layers with exact localization. As not all five human skin specimen had enough frozen sections for all skin layers, thus, the number variable in the heatmap of Fig. 2a. However, at least three samples were included in each skin regions (n≥3).
The modules 1-6 have been labeled in the heatmap to associate the regions with the descriptions on the right side.
In Fig. 2c, we have added the dashed lines, redone the staining experiments of EGFR and COL4A1, and added the arrows to indicate areas of specific staining. In 3a, what is the staining shown under "Treponema pallidum"? The staining method is not described. Also, the coloration in the lower right panel of 3a does not match that of the low magnification view. Scale bars should be used consistently and labeled. In 3b legend, SSP-GS should be described as the stratum granulosum-spinosum layers rather than stratum corneum. For 3d legend, the term "gradually" does not fit here. In 3e, the NC and SSP groups should be more clearly separated in the figure.
Answer: Many thanks for the reviewer's suggestion. We have corrected the coloration labels in Fig.3a. In order to present the expression levels of KRT10 and KRT14 better, the DAPI (blue) wasn't shown in low magnification view.
The scale bars have been labeled with the images in the revised manuscript.
We have corrected the description of SSP-GS as "granular-spinous" in Fig. 3b legend.
The NC and SSP groups have been clearly separated in Fig. 3e by a black line of dashes.  l. 248 and elsewhere. expression rather than expressions.
Answer: Many thanks for the reviewer's suggestion.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): Authors addressed all of my concerns; however, a few minor points are required to be revised before the manuscript can be accepted.
1. Quantification data for immunostaining analyses shown in Fig.4g, 4k, 4o, 5a, 5c, 5d and 7c. What are those numbers at the corner of Fig. 4g, 4k, 4o, 5a, 5c, 5d, 7b and 7c referred to? Numbers or percentage of positive cells per image or per spheroid? Given that cell numbers could vary a lot from one image to another, it is more accurate to use percentage to present the quantification data. Please define it in the figure legends accordingly. Fig. 3f should indicate basement membrane by dotted lines as those shown in Fig. 4a. Even though the authors had changed a new image, the staining for TGFBI in Fig. 3f remains highly positive to the epidermis. It seems overlapping with KRT14 staining. Please double confirm it by performing new staining. Fig. 4h? Almost 100% of cells with E-cadherin staining at cell-cell junction. It is surprising to see only 20-30 presented in the images unless the numbers are referred to the cell number. If this is the case, it should be re-calculated and presented as percentage.

Please explain how the authors quantified E-cad+ cells shown in
4. As for Fig. 5, it will be helpful if the Z-stack images that are shown in Figure 2 (rebuttal letter) can be provided in the supplementary figures.

5.
As for western blotting analysis, both phosphorylated and total proteins should be provided for assessment. p-GSK3b-S9 needs to be provided in Fig. 4k. Total GSK3b protein is missing in Fig. S6i. p-b-catenin S33/37/41 is not provided in Fig. S6j. The loading control for nuclear protein, e.g. laminin, is required for Fig. 4l.
Reviewer #2 (Remarks to the Author): I thank the authors for responding to my question regarding randomization, although I wasn't clear enough about what I was asking. Were the mass spec samples run at the same time, and was a randomized run order used? Were the samples all run at different times? The response sounds as though samples from each individual patient, regardless of condition, were all processed and analyzed at completely different times. This could impact results for quantification, especially if all controls were run and then all with secondary syphilis (for example). If samples from all patients were run at different points in time, were the different layers analyzed in the same order every time or were they randomized? All of these factors could impact the results. Normalization helps to some extent, but I'm not sure if it would completely compensate for samples being analyzed at completely different points in time and in the exact same order every time.
Reviewer #3 (Remarks to the Author): The manuscript represents a detailed, major work on the distribution of proteins in the skin using a combination of mass spectroscopy, immunolabeling, and other approaches. It has been revised extensively from its prior version, and it appears that the most of the reviewers' comments have been addressed effectively. Very minor grammatical issues remain, including the improper use of the word "contract" (construct?) on l. 150 and the common use of the word "expressions" where "expression" is appropriate. We performed new staining of TGFBI and KRT14 in health and SSP skins and updated the staining results in Fig. 3f. TGFBI is mainly expressed around the basal stem cell layer (BL layer) and at the bottom of the BL layer, indicating that TGFBI is mainly located around the basement membrane after secreted by cells. KRT14 is also mainly expressed in the basal stem cell layer, and secreted outside the cells. Therefore, TGFBI and KRT14 are almost co-expression in the skin. In addition, the basement membrane has been indicated with dotted lines in Fig. 3f.  Fig. 4h? Almost 100% of cells with E-cadherin staining at cell-cell junction. It is surprising to see only 20-30 presented in the images unless the numbers are referred to the cell number. If this is the case, it should be re-calculated and presented as percentage.

Please explain how the authors quantified E-cad+ cells shown in
Answer: Many thanks for the editor's suggestion. The fluorescence intensity was used to quantify the protein expression level by Image J analysis software. For ECAD single channel pictures, the threshold was adjusted first; then the appropriate threshold algorithm (Default) was selected and applied to all pictures in different groups. Next, the parameters were set to ensure that the measured value is just for the proteins expressed in the specific region. Finally, the percentage of average fluorescence intensity of ECAD (%) is calculated by the following formula: Average fluorescence intensity (Mean) % = Sum of fluorescence intensity of the region (IntDen) / Area of the region (Area) *100%..
The numbers in Fig. 4f represented the percentage of the average fluorescence intensity of the specific protein (fluorescent channel); we forgot to add the percent symbol (%) in the previous figures. In this revised version, we corrected this mistake and added the percent symbol (%) for quantified data in  Answer: Many thanks for the editor's suggestion.
In this revised manuscript, several representative Z-stack images of 3D spheroids have been provided as Supplementary Fig. 7a to support the results in Fig. 5.

Reviewer #2 (Remarks to the Author):
I thank the authors for responding to my question regarding randomization, although I wasn't clear enough about what I was asking. Were the mass spec samples run at the same time, and was a randomized run order used? Were the samples all run at different times? The response sounds as though samples from each individual patient, regardless of condition, were all processed and analyzed at completely different times. This could impact results for quantification, especially if all controls were run and then all with secondary syphilis (for example). If samples from all patients were run at different points in time, were the different layers analyzed in the same order every time or were they randomized? All of these factors could impact the results. Normalization helps to some extent, but I'm not sure if it would completely compensate for samples being analyzed at completely different points in time and in the exact same order every time.
Answer: Many thanks for the editor's suggestion. All skin tissue samples prepared for mass spectrometer (MS) were processed simultaneously. To avoid the error caused by different MS instrument, all peptides samples were run use the same HF-X Orbitrap MS in a sequential order at different time. The tissue samples preparation and peptide mixtures analysis by MS were processed by two different investigators. Both investigators were kept blinded to the group (control or secondary syphilis) and layer (skin layers) information. Thus, all samples were analyzed using exactly the same procedures and in randomized orders on MS instrument.
For the quality control of the performance of MS platform, the HEK293T cell lysate was measured every two days as the quality-control standard sample. The HEK293T standard sample was digested and analyzed using the same method, condition, and MS instrument as the skin samples. In the whole MS experiment procedures, six HEK293T standard samples (Sample 1-6) were analyzed and generated six MS datasets. As shown in Figure 1, the six replicated standard samples identified similar protein numbers (Figure 1a), and more than 90% of the proteins (4722, Figure 1b) were identified by all samples. A pairwise spearman correlation coefficient was calculated for protein quantification of all quality-control samples, the average correlation coefficient among the standards was 0.98. All these results demonstrated the consistent stability of the mass spectrometry platform. Also, we have added the detailed information about this quality control process in "MS analysis" section in Methods.

Reviewer #3 (Remarks to the Author):
The manuscript represents a detailed, major work on the distribution of proteins in the skin using a combination of mass spectroscopy, immunolabeling, and other approaches. It has been revised extensively from its prior version, and it appears that the most of the reviewers' comments have been addressed effectively. Very minor grammatical issues remain, including the improper use of the word "contract" (construct?) on l. 150 and the common use of the word "expressions" where "expression" is appropriate.
Answer: Many thanks for the editor's suggestion. We have corrected the word "contract" to "construct". We have taken the reviewer's suggestion and use the common use of the word "expression".